Methods for treating diseases associated with respiratory viruses

ABSTRACT

The present disclosure provides a method of treating a disease associated with a respiratory virus. The method comprises administering an effective amount of a pharmaceutical composition prepared by removing albumin from a solution of a human serum albumin composition and/or comprising a diketopiperazine with amino acid side chains of aspartic acid and alanine (DA-DKP), such as a low molecular weight fraction of human serum albumin. The present disclosure also provides a pharmaceutical product as well as a kit comprising DA-DKP.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/482,144, filed Sep. 22, 2021; which is a continuation of U.S. patentapplication Ser. No. 17/211,553, filed Mar. 24, 2021, and issued on Sep.28, 2021 as U.S. Pat. No. 11,129,878; which claims the benefit of U.S.Provisional Patent Application No. 62/994,171, filed Mar. 24, 2020; thedisclosures of each of which are incorporated herein by reference intheir entirety.

FIELD

The present disclosure relates to a method of treating diseasesassociated with respiratory viruses, also referred to herein as viralrespiratory diseases. The method comprises administering an effectiveamount of a pharmaceutical composition prepared by removing albumin froma solution of a human serum albumin composition and/or comprising adiketopiperazine with amino acid side chains of aspartic acid andalanine (DA-DKP). The present disclosure also provides a pharmaceuticalproduct comprising DA-DKP, in some embodiments in a nebulized form.

BACKGROUND

Common viral respiratory diseases are illnesses caused by a variety ofviruses that have similar traits and affect the upper respiratory tract.The viruses involved may be the coronaviruses, influenza viruses,respiratory syncytial virus (RSV), parainfluenza viruses, or respiratoryadenoviruses. Coronaviruses are a group of related viruses that causediseases in mammals and birds. In humans, coronaviruses causerespiratory tract infections that can be mild, such as some cases of thecommon cold (among other possible causes, predominantly rhinoviruses),and others that can be lethal, such as SARS, MERS, and COVID-19.Symptoms in other species vary: in chickens, they cause an upperrespiratory tract disease, while in cows and pigs they cause diarrhea.

Diketopiperazines have been reported to exhibit a variety of biologicalactivities. See, e.g., U.S. Pat. No. 4,289,759 (immunoregulatoryagents), 4,331,595 (immunoregulatory agents), 4,940,709 (PAFantagonists), 5,700,804 (inhibitors of plasminogen activator inhibitor),5,750,530 (inhibitors of plasminogen activator inhibitor) and 5,990,112(inhibitors of metalloproteases); PCT publication nos. WO 97/36888(inhibitors of farnesyl-protein transferase) and WO 99/40931 (treatmentof central nervous system injury); EP Patent No. 0043219(immunoregulatory agents); Japanese patent application nos. 63 290868(PAF antagonists) and 31 76478 (immunosuppressive agents); and Shimazakiet al., Chem. Pharm. Bull., 35(8), 3527-3530 (1987) (PAF antagonists),Shimazaki et al., J. Med. Chem., 30, 1709-1711 (1987) (PAF antagonists),Shimazaki et al., Lipids, 26(12), 1175-1178 (1991) (PAF antagonists),Yoshida et al., Prog. Biochem. Pharmacol., 22, 68-80 (1988) (PAFantagonists), Alvarez et al., J. Antibiotics, 47(11), 1195-1201 (1994)(inhibitors of calpain).

SUMMARY

In a first aspect, the present disclosure provides a method of treatingor preventing one or more symptoms of a viral respiratory disease in apatient, comprising administering to the patient a pharmaceuticalcomposition prepared by removing albumin from a solution of a humanserum albumin composition.

In a second aspect, the present disclosure provides a method of treatingor preventing a viral respiratory disease in a patient, comprisingadministering to the patient a pharmaceutical composition comprisingDA-DKP.

In a third aspect, the present disclosure provides a method of treatingor preventing inflammation associated with a viral respiratory diseasein a patient, comprising administering to the patient a pharmaceuticalcomposition prepared by removing albumin from a solution of a humanserum albumin composition.

In a fourth aspect, the present disclosure provides a method of treatingor preventing inflammation associated with a viral respiratory diseasein a patient, comprising administering to the patient a pharmaceuticalcomposition comprising DA-DKP.

In some embodiments the viral respiratory disease is selected from thegroup consisting of Severe Acute Respiratory Distress Syndrome (SARS),Middle East Respiratory Syndrome (MERS), COVID-19, and a viral infectionassociated with asthma, pneumonia, bronchitis and/or tuberculosis. Insome embodiments, the viral respiratory disease is COVID-19.

In some embodiments the viral respiratory disease is caused by a virusselected from group consisting of a coronavirus, an influenza virus,respiratory syncytial virus (RSV), a parainfluenza virus, and arespiratory adenovirus. The virus is, in some embodiments, selected fromthe group consisting of SARS-Coronavirus-2 (SARS-CoV-2), SARS-associatedcoronavirus (SARS-CoV), and Middle East Respiratory Syndrome Coronavirus(MERS-CoV). In some aspects the method of Claim 8, wherein the virus isSARS-Coronavirus-2 (SARS-CoV-2).

In some embodiments the patient has or is at risk of developinginflammation of a tissue selected from the group consisting of lung,brain, heart, kidney, blood vessel, skin, and nerve. In someembodiments, the tissue is lung.

In some embodiments the patient has or is at risk of developing asymptom selected from the group consisting of acute respiratory distresssyndrome (ARDS), acute lung injury (ALI), interstitial lung disease,pulmonary fibrosis, pneumonia, and reactive airway disease syndrome.

In some embodiments the patient has or is at risk of developing asymptom selected from the group consisting of fatigue, shortness ofbreath or difficulty breathing, low exercise tolerance, low blood oxygensaturation, cough, sore throat, stuffy or runny nose, joint pain, chestpain, tightness or discomfort, muscle pain, muscle weakness, fever,heart palpitations, difficulty thinking and/or concentrating, anddepression.

In some embodiments the patient has experienced the symptom at leastfour weeks, at least one month, at least two months, or at least threemonths.

In some embodiments the administering results in an outcome selectedfrom the group consisting of reduced ventilator time, reduced mortality,improvement in oxygenation parameters, reduced time to resolution of oneor more respiratory symptoms, improved pulmonary function, andcombinations thereof.

In certain embodiments, after the administration, the patient achievesimprovement on the World Health Organization COVID-19 ordinal scale ofat least 4, at least 3, at least 2, or at least 1 (World HealthOrganization. (2020). WHO R&D Blueprint: novel Coronavirus: COVID-19Therapeutic Trial Synopsis, Feb. 18, 2020, Geneva, Switzerland).

In some embodiments the patient has respiratory distress, and in certainembodiments the patient requires supplemental oxygen.

In some embodiments the pharmaceutical composition reduces vascularpermeability in the patient, increases the production of lungprostaglandins in the patient, and/or decreases the amount or activityof one or more lung inflammatory signaling proteins in the patient. Incertain aspects the one or more lung inflammatory signaling proteinsis/are selected from the group consisting of TNF-alpha, IL6 and CXCL10.

In some embodiments the composition is administered in a form suitablefor administration to the lung. In some embodiments composition isadministered in a nebulized form.

In some embodiments the composition is administered at a dose of 8milliliters, and in some embodiments the composition is administeredquater in die. In some embodiments the composition is administered at adose of 8 milliliters quater in die. In some embodiments the compositionis administered for five days.

In some embodiments the composition is administered in an aerosolizedform.

In some embodiments the composition is administered intravenously. Insome embodiments the composition is administered at a dose of 250 cubiccentimeters, and in some embodiments the composition is administered bisin die. In certain embodiments the composition is administered at a doseof 250 cubic centimeters bis in die. In some embodiments the compositionis administered for five days.

In some embodiments the composition comprises DA-DKP, and in someembodiments the composition further comprises N-acetyl-tryptophan (NAT),caprylic acid, caprylate or combinations thereof. In some embodimentsthe DA-DKP is in a composition prepared by removing albumin from asolution of a human serum albumin composition. In some embodimentsremoving the albumin comprises treating the human serum albumincomposition by a separation method selected from the group consisting ofultrafiltration, sucrose gradient centrifugation, chromatography, saltprecipitation, and sonication. In some embodiments the removingcomprises passing the human serum albumin composition over anultrafiltration membrane with a molecular weight cut off that retainsthe albumin, and wherein the resulting filtrate comprises DA-DKP. Incertain aspects the ultrafiltration membrane has a molecular weightcutoff of less than 50 kDa, less than 40 kDa, less than 30 kDa, lessthan 20 kDa, less than 10 kDa, less than 5 kDa or less than 3 kDa.

In a fifth aspect, the present disclosure provides a pharmaceuticalproduct comprising a DA-DKP-containing composition, formulated foradministration in some embodiments by nebulization or aerosolization,and in some embodiments formulated for intravenous administration. Insome embodiments the DA-DKP is prepared by removing albumin from asolution of a human serum albumin composition. In certain embodimentsremoving the albumin comprises treating the human serum albumincomposition by a separation method selected from the group consisting ofultrafiltration, sucrose gradient centrifugation, chromatography, saltprecipitation, and sonication. In some embodiments the removingcomprises passing the human serum albumin composition over anultrafiltration membrane with a molecular weight cut off that retainsthe albumin, and wherein the resulting filtrate comprises DA-DKP. Insome embodiments the ultrafiltration membrane has a molecular weightcutoff of less than 50 kDa, less than 40 kDa, less than 30 kDa, lessthan 20 kDa, less than 10 kDa, less than 5 kDa or less than 3 kDa. Incertain aspects the DA-DKP-containing composition further comprises NAT,caprylic acid, caprylate and combinations thereof.

Some embodiments include of the pharmaceutical composition include apharmaceutically-acceptable carrier.

In a sixth aspect, the present disclosure provides a kit comprising apharmaceutical product comprising a DA-DKP-containing compositionformulated for administration by nebulization or aerosolization. In someembodiments the kit comprising a pharmaceutical product comprising aDA-DKP-containing composition formulated for intravenous administration.In certain embodiments the DA-DKP is prepared by removing albumin from asolution of a human serum albumin composition.

DETAILED DESCRIPTION

The present disclosure generally relates to a method of treating a viralrespiratory disease. The treatment comprises administering an effectiveamount of a pharmaceutical composition prepared by removing albumin froma solution of a human serum albumin composition and/or comprisingaspartyl-alanyl diketopiperazine (DA-DKP) to an animal having a needthereof.

The present disclosure also relates to a method of reducing lunginflammation associated with a viral infection in an animal in needthereof. The method comprises administering an effective amount of apharmaceutical composition comprising DA-DKP to an animal having a needthereof.

The present disclosure further relates to a method of reducing vascularpermeability in lungs of an animal in need thereof. The method comprisesadministering an effective amount of a pharmaceutical compositioncomprising DA-DKP to an animal having a need thereof.

The present disclosure also relates to a method to up regulate theproduction of lung prostaglandins in an animal in need thereof. Themethod comprises administering an effective amount of a pharmaceuticalcomposition comprising DA-DKP to an animal having a need thereof.

The present disclosure further relates to a method to down regulate oneor more lung inflammatory signaling proteins in a subject in needthereof. The method comprises administering an effective amount of apharmaceutical composition comprising DA-DKP to an animal having a needthereof. In one aspect, the lung inflammatory signaling protein isTNF-alpha, interleukin-6 (IL6) and C-X-C motif chemokine ligand 10(CXCL10).

The present disclosure also provides for a pharmaceutical productcomprising a DA-DKP composition formulated for administration to thelungs, including a nebulized form or aerosolized form. The DA-DKP of theproduct may be prepared by removing albumin from a solution of humanserum albumin.

The present disclosure also provides for kit comprising a DA-DKPcomposition formulated for administration to the lungs, including anebulized form or aerosolized form.

DA-DKP has multiple anti-inflammatory and immune modulating effectsincluding inhibition of multiple pro-inflammatory cytokines, chemokinesand signaling molecules at the transcription level, inhibition of themigration and adhesion of T-cells and monocytes, activity at theG-coupled protein receptor level, activity on actin-dependentcytoskeletal events, and reduction in vascular permeability andinhibition of inflammation induced by platelet activating factor, amongother effects. Because inflammation is exacerbated by, or involves,activated T-cells, the composition of the present disclosure comprisingDA-DKP can be used to treat lung inflammatory diseases and/orinflammation related to viral respiratory diseases and/or to preventlung inflammation and lung inflammatory diseases related to viralrespiratory diseases. Thus, one embodiment of the present disclosure isa method of reducing inflammation caused by a viral respiratory diseasein an individual.

The pharmaceutical composition comprising DA-DKP can be prepared byremoving albumin from a solution of human serum albumin as disclosedherein.

A viral respiratory disease is an illness caused by a virus and affectsthe respiratory tract. Such viral respiratory diseases can includeSevere Acute Respiratory Distress Syndrome (SARS), Middle EastRespiratory Syndrome (MERS), COVID-19, and viral infection associatedwith asthma, pneumonia, bronchitis and/or tuberculosis. Viruses that cancause one or more viral respiratory diseases include coronaviruses,influenza viruses, respiratory syncytial virus (RSV), parainfluenzaviruses, and respiratory adenoviruses. Coronaviruses includeSARS-Coronavirus-2 (SARS-CoV-2), SARS-associated coronavirus (SARS-CoV),and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Coronavirusinfections and other viral infections can cause acute respiratorydistress syndrome (ARDS), acute lung injury (ALI), interstitial lungdisease, pulmonary fibrosis, pneumonia, and reactive airway diseasesyndrome. Coronavirus infections and other viral infections can causeinflammation in tissues such as lung, brain, heart, kidney, bloodvessel, skin, and nerve. Coronavirus infections and other viralinfections can cause symptoms such as fatigue, shortness of breath ordifficulty breathing, low exercise tolerance, low blood oxygensaturation, cough, sore throat, stuffy or runny nose, joint pain, chestpain, tightness, or discomfort, muscle pain, muscle weakness, fever,heart palpitations, difficulty thinking and/or concentrating, anddepression.

COVID-19 infection is a respiratory illness caused by the novelcoronavirus SARS-COV-2 and has been classified as a pandemic with noknown cure to date. COVID-19 is detected and diagnosed with a laboratorytest. The primary symptoms of COVID-19 infection include mild symptomssuch as fever, cough, chills, muscle pain, headache, gastrointestinalsymptoms, and the loss of taste or smell. Once infected, the virus movesdown a patient's respiratory tract, where the lungs may become inflamed,making breathing difficult and sometimes requiring supplemental oxygenin the more severe cases of the disease.

Respiratory symptoms after a COVID-19 infection include shortness ofbreath, cough, chest discomfort, low exercise tolerance and low oxygensaturation, all of which point to potential inflammation relatedcomplication sequalae. Infiltrating or resident cells in the immunesystem (e.g., macrophages, peripheral blood mononuclear cells, etc.) maybe responsible for the development of these respiratory long-termconsequences. Chronic or prolonged inflammation of the lungs mayberesponsible for a myriad of respiratory signs and symptoms experiencedby patients after a COVID-19 infection. Chest x-rays and CT scans revealdisturbing patterns of perhaps extensive fibrosis and potential loss ofelasticity and oxygen diffusion capacity.

The SARS-Cov-2 virus transmits through the respiratory system and cancause a severe dysregulation of the immune response and damage in thelungs. Chronic, prolonged inflammation of the lungs may be responsiblefor a myriad of continuing respiratory signs and symptomspost-infection, including cough, shortness of breath, chest discomfort,low exercise tolerance and low blood oxygen saturation. The continuedhyperinflammatory state is thought to lead to prolonged clinicalcomplications, and treatment with immunomodulators at this later pointin the disease is more effective than anti-viral treatment.

Inflammation associated with COVID-19 may trigger even more severecomplications including pneumonia, acute lung injury (ALI) and/or acuterespiratory distress syndrome (ARDS), which is a leading cause ofmortality in COVID-19. ARDS is associated with widespread inflammationin the lungs. The underlying mechanism of ARDS involves diffuse injuryto cells which form the barrier of the microscopic air sacs (alveoli) ofthe lung, surfactant dysfunction, and activation of the immune system.The fluid accumulation in the lungs associated with ARDS is partiallyexplained by vascular leakage due to inflammation.

As disclosed herein, the composition of the present disclosure, in anaqueous solution that can be delivered through nebulization, can be usedfor suppressing inflammation in the lungs, thus making it a therapy forARDS. An important aspect of ARDS, triggered by COVID-19, is an initialrelease of chemical signals and other inflammatory mediators secreted bylung epithelial and endothelial cells. Neutrophils and someT-lymphocytes migrate into the inflamed lung tissue and contribute tothe amplification/deterioration of ARDS. A decrease in the production oflipid mediators of inflammation (prostaglandins) may impair theresolution of inflammation associated with ARDS (Fukunaga, et. al.,Cyclooxygenase 2 Plays a Pivotal Role in the Resolution of Acute LungInjury. Journal of Immunology 2005; 174:5033-5039; Gao et al J Immunol2017; 199:2043-2054).

The World Health Organization (WHO)'s Clinical Care for Severe AcuteRespiratory Infection: COVID-19 Adaptation recommends early interventionwith supplemental oxygen for COVID-19 patients with low blood oxygensaturation (SpO₂) beginning with the least invasive modality possible(e.g. hand-held oxygen source) and moving to more invasive modalities(e.g. bilevel positive airway pressure [BiPAP] and/or non-invasiveventilation (NIV)) as severity increases. Treatment during earlyintervention for COVID-19 patients with respiratory distress requiresmonitoring of respiratory function with treatment responsive to diseaseprogression. The CDC recommends following the guidelines for treatmentof COVID-19 patients with hypoxia in Surviving Sepsis Campaign:Guidelines on the Management of Critically Ill Adults with CoronavirusDisease 2019 (COVID-19).

Patients who fail to respond to less-invasive treatment are at a highrisk of developing ARDS, a rapidly progressive disease characterized bywidespread inflammation in the lungs that results in flooding of thelungs' microscopic air sacs, which are responsible for the exchange ofgases such as oxygen and carbon dioxide with capillaries in the lungs.Additional common findings in ARDS include partial collapse of the lungs(atelectasis) and low levels of oxygen in the blood (hypoxemia). Theclinical syndrome is associated with pathological findings includingpneumonia and diffuse alveolar damage, the latter of which ischaracterized by diffuse inflammation of lung tissue. The triggeringinsult to the tissue usually results in an initial release of chemicalsignals and other inflammatory mediators secreted by local epithelialand endothelial cells.

ARDS impairs the lungs' ability to exchange oxygen and carbon dioxide.The underlying mechanism of ARDS involves diffuse injury to cells thatform the barrier of the microscopic air sacs of the lungs, surfactantdysfunction, activation of the immune system, and dysfunction of thebody's regulation of blood clotting.

Diagnosis of ARDS is based on the 2012 Berlin definition:

-   -   lung injury of acute onset, within 1 week of an apparent        clinical insult and with progression of respiratory symptoms    -   bilateral opacities on chest imaging (chest radiograph or CT)        not explained by other lung pathology (e.g. effusion, lobar/lung        collapse, or nodules)    -   respiratory failure not explained by heart failure or volume        overload    -   decreased ratio of partial pressure arterial oxygen (PaO₂) to        fraction of inspired oxygen (FiO₂) of less than or equal to 300        mm Hg despite a positive end-expiratory pressure (PEEP) of more        than 5 cm H₂O.

The severity of ARDS is defined by the Berlin definition as:

-   -   mild ARDS: 201-300 mmHg (≤39.9 kPa)    -   moderate ARDS: 101-200 mmHg (≤26.6 kPa)    -   severe ARDS: ≤100 mmHg (≤13.3 kPa)

There are no approved treatments for ARDS, and standard of care (SOC) issupportive management.

Furthermore, COVID-19 can cause prolonged symptoms even after the viralinfection has seemingly been cleared. These prolonged effects arereferred to a post-COVID, post-acute COVID, post-acute sequelae ofSARS-CoV-2 infection (PASC), chronic COVID syndrome (CCS), and long-haulCOVID. The many symptoms can include fatigue, shortness of breath ordifficulty breathing, low exercise tolerance, low blood oxygensaturation, cough, sore throat, stuffy or runny nose, joint pain, chestpain, tightness or discomfort, muscle pain, muscle weakness, fever,heart palpitations, difficulty thinking and/or concentrating, anddepression. Symptoms can also include breathlessness, myalgia, anxiety,extreme fatigue, low mood, sleep disturbances or difficulty sleeping,and memory impairment. Other neurologic symptoms include brain fog(non-specific cognitive problems), headache, numbness or tingling,dysgeusia, dizziness, pain, blurred vision, tinnitus, and loss of taste(ageusia) or smell (anosmia). Symptoms can last for a month or more,including at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,at least 12 months, or longer. The continued hyperinflammatory state isthought to lead to prolonged clinical complications, and treatment withimmunomodulators at this later point in the disease is more effectivethan anti-viral treatment.

In addition to lung symptoms and long COVID, SARS-CoV-2 infection canlead to damage to other organs, including the heart and kidneys.SARS-CoV-2 infects endothelial cells and also leads to systemicinflammation, causing vasculopathy that affects widespread parts of thebody. The vasculopathy or other phenomena can cause damage to the heart(myocarditis or arrhythmia), kidneys (acute kidney injury, chronickidney disease, or renal failure), liver (liver dysfunction), bloodvessels (bleeding and blood clots), skin (Kawasaki-like syndrome, rash,hair loss, and urticarial, vesicular, purpuric, and papulosquamouslesions), digestive system (anorexia, nausea, vomiting, diarrhea, andabdominal pain), brain (described above), and nerves (symptoms describedabove and cerebrovascular disease, ataxia, seizure, vision impairment,and nerve pain). Other symptoms include lymphopenia, hypoxia, bloodhypercoagulability, multi-organ failure, sepsis, and septic shock.

Conventional pharmaceutical therapies for viral respiratory diseaseinclude anti-viral compositions such as amantadine and ribavin.

In any of the methods and compositions disclosed herein, forms foradministration of the composition of the present disclosure includenebulized form, aerosolized form, sprays, drops, and powders. The activeingredient may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required. In some embodiments, the form foradministration is a sterile liquid that is administered as a nebulizedliquid form or intravenously.

Aerosol (inhalation) delivery can be performed using methods standard inthe art. Carriers suitable for aerosol delivery are described herein.Devices for delivery of aerosolized formulations include, but are notlimited to, pressurized metered dose inhalers (MDI), dry powder inhalers(DPI), and metered solution devices (MSI), and include devices that arenebulizers and inhalers.

The composition of the present disclosure comprising DA-DKP can beadministered to an animal by any suitable route of administration to thelungs, including nasal, intratracheal, bronchial, direct instillationinto the lung, inhaled and oral.

“Treat” is used herein to mean to reduce (wholly or partially) thesymptoms, duration or severity of a disease. In accordance with thepresent state of the art, treat typically does not mean to cure. Asprovided for herein, in any of the methods disclosed herein treatingincludes that the animal experiences an outcome including, but notlimited to, reduced ventilator time, reduced mortality, improvement inoxygenation parameters and combinations thereof.

The pharmaceutical composition comprising DA-DKP of the presentdisclosure is administered to an animal in need of treatment. In someembodiments, the animal is a mammal, such as a rabbit, goat, dog, cat,horse or human. In some embodiments, the animal in need of treatment isa human. Effective dosage amounts may vary with the severity of thedisease or condition, the route(s) of administration, the duration ofthe treatment, the identity of any other drugs being administered to theanimal, the age, size and species of the animal, the discretion of theprescribing health care provider, and like factors known in the medicaland veterinary arts.

The composition of the present disclosure may be a pharmaceuticalsolution having a DA-DKP concentration range with a lower endpoint ofabout 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110μM, about 120 μM, about 130 μM, about 140 μM, about 150 μM, about 160μM, about 170 μM, about 180 μM, about 190 μM, about 200 μM, about 210μM, about 220 μM, about 230 μM, about 240 μM, about 240, about 250 μM,about 260 μM, about 270 μM, about 280 μM, about 290 μM, about 300 μM,about 310, about 320 μM, about 330 μM, about 340 μM, about 350 μM, about360 μM, about 370 μM, about 380 μM, about 390 μM, or about 400 μM. Thecomposition of the present disclosure may be a pharmaceutical solutionhaving a DA-DKP concentration range with an upper endpoint of about 600μM, about 580 μM, about 570 μM, about 560 μM, about 550 μM, about 540μM, about 530 μM, about 520 μM, about 510 μM, about 500 μM, about 490μM, about 480 μM, about 470 μM, about 460 μM, about 450 μM, about 440μM, about 430 μM, about 420 μM, about 410 μM, about 400 μM, about 390μM, about 380 μM, about 370 μM, about 360 μM, about 350, about 340 μM,about 330 μM, about 320 μM, about 310 μM, about 300 μM, about 290 μM,about 280, about 270 μM, about 260 μM, about 250 μM, about 240 μM, about230 μM, about 220 μM, about 210 μM, or about 200 μM.

An effective amount of DA-DKP in the composition of the presentdisclosure for treating a viral respiratory disease or condition can bea range with a lower endpoint of about 10 μg, about 15 μg, about 20 μg,about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg,about 80 μg, about 85 μg, about 90 μg, about 95 μg, about 100 μg, about110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about160 μg, about 170 μg, about 180 μg, about 190 μg, about 200 μg, about210 μg, about 220 μg, about 230 μg, about 240 μg, about 250 μg, about260 μg, about 270 μg, about 280 μg, about 290 μg, about 300 μg, about310 μg, about 320 μg, about 330 μg, about 340 μg, about 350 μg, about360 μg, about 370 μg, about 380 μg, about 390 μg, about 400 μg, about425 μg, about 450 μg, about 475 μg or about 500 μg. In addition, aneffective amount of DA-DKP in the composition of the present disclosurefor treating a viral respiratory disease or condition can be a rangewith upper endpoint of about 500 μg, about 490 μg, about 480 μg, about470 μg, about 460 μg, about 450 μg, about 440 μg, about 430 μg, about420 μg, about 410 μg, about 400 μg, about 390 μg, about 380 μg, about370 μg, about 360 μg, about 350 μg, about 340 μg, about 330 μg, about320 μg, about 310 μg, about 300 μg, about 290 μg, about 280 μg, about270 μg, about 260 μg, about 250 μg, about 240 μg, about 230 μg, about220 μg, about 210 μg, about 200 μg, about 190 μg, about 180 μg, about170 μg, about 160 μg, about 150 μg, about 140 μg, about 130 μg, about120 μg, about 110 μg, about 100 μg, about 90 μg, about 80 μg, about 70μg, about 60 μg, about 50 μg, about 40 μg, about 30 μg, or about 20 μg.

Different doses of the disclosed compositions can be used with differentroutes of administration. Administration to the lung, for example bynebulizer, can involve doses of about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, or about 20 milliliters. Intravenous administration can involvedoses of about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, about 95, about 100, about 105, about 110,about 115, about 120, about 125, about 130, about 135, about 140, about145, about 150, about 155, about 160, about 165, about 170, about 175,about 180, about 185, about 190, about 195, about 200, about 205, about210, about 215, about 220, about 225, about 230, about 235, about 240,about 245, about 250, about 260, about 270, about 275, about 280, about290, about 300, about 310, about 320, about 330, about 340, about 350,about 360, about 370, about 375, about 380, about 390, about 400, about425, about 450, about 475, about 500, about 525, about 550, about 575,about 600, about 650, about 700, about 750, about 800, about 850, about900, about 950, about 1000, about 1100, about 1200, about 1300, about1400, or about 1500 milliliters (or cubic centimeters). The disclosedcompositions can be administered 1, 2, 3, 4, 5, 6, 7, 8, or more timesper day for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.Disclosed compositions can also be administered for 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 weeks, and/or for 4, 5, 6, 7, 8, 9, 10, 11, or 12 monthsor more.

While it is possible for a compound of the present disclosure, such asDA-DKP, to be administered alone, in many embodiments the compound isadministered as a pharmaceutical formulation (composition). Thepharmaceutical compositions of the present disclosure comprise acompound or compounds of the present disclosure as an active ingredientin admixture with one or more pharmaceutically-acceptable carriers and,optionally, with one or more other compounds, drugs or other materials.In some embodiments, the compound is DA-DKP. Each carrier isadvantageously “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the animal.Pharmaceutically-acceptable carriers are well known in the art.Regardless of the route of administration selected, the compounds of thepresent disclosure are formulated into pharmaceutically-acceptabledosage forms by conventional methods known to those of skill in the art.See, e.g., Remington's Pharmaceutical Sciences (Easton, Pa.: Mack Pub.Co, 1965. Print; 23^(rd) Ed. (2020) ISBN: 9780128200070).

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present disclosureinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for nebulization, immediately prior to use.

Kits comprising the pharmaceutical products of the present disclosureare also provided. The kits can comprise a DA-DKP composition formulatedfor administration to the lung including a nebulized form and/or anaerosolized form. The DA-DKP can be prepared as described herein, suchas by removing albumin from a solution of a human albumin composition.The kits may contain unit-dose or multi-dose sealed containers, forexample, ampoules and vials, and may be stored in a lyophilizedcondition requiring only the addition of the sterile liquid carrier, forexample water, immediately prior to use. The kits may also be stored ina condition, wherein the contents are ready for direct use or injection.

The composition of the present disclosure may further compriseN-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinationsthereof. In some embodiments, the composition comprises NAT.Compositions of the present disclosure having NAT, caprylic acid,caprylate or combinations thereof have a concentration range with alower endpoint of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM,about 17 mM, about 18 mM, about 19 mM, or about 20 mM. In addition,compositions of the present disclosure having NAT, caprylic acid,caprylate or combinations thereof have a concentration range with anupper endpoint of about 40 mM, about 39 mM, about 38 mM, about 37 mM,about 36 mM, about 35 mM, about 34 mM, about 33 mM, about 32 mM, about31 mM, about 30 mM, about 29 mM, about 28 mM, about 27 mM, about 26 mM,about 25 mM, about 24 mM, about 23 mM, about 22, or about 21 mM. In someembodiments, the concentration range is about 4 mM to about 20 mM.

In addition, the composition of the present disclosure can also comprisea second drug such as an anti-viral, an antibiotic, chloroquine,hydroxychloroquine, known drugs for treating pneumonia, an analgesic(such as lidocaine or paracetamol), an anti-inflammatory (such ascorticosteroids, such as dexamethasone and betamethasone, non-steroidanti-inflammatory drugs (NSAIDs), ibuprofen, naproxen), and/or othersuitable drugs.

Methods of making diketopiperazines, such as DA-DKP, are well known inthe art, and these methods may be employed to synthesize thediketopiperazines of the present disclosure. See, e.g., U.S. Pat. Nos.4,694,081, 5,817,751, 5,990,112, 5,932,579 and 6,555,543, US PatentApplication Publication Number 2004/0024180, PCT applications WO96/00391 and WO 97/48685, and Smith et al., Bioorg. Med. Chem. Letters,8, 2369-2374 (1998).

For instance, diketopiperazines, such as DA-DKP, can be prepared byfirst synthesizing dipeptides. The dipeptides can be synthesized bymethods well known in the art using L-amino acids, D-amino acids or acombination of D- and L-amino acids. In some embodiments solid-phasepeptide synthetic methods are employed. Of course, dipeptides are alsoavailable commercially from numerous sources, including DMI SynthesisLtd., Cardiff, UK (custom synthesis), Sigma-Aldrich, St. Louis, Mo.(primarily custom synthesis), Phoenix Pharmaceuticals, Inc., Belmont,Calif. (custom synthesis), Fisher Scientific (custom synthesis) andAdvanced ChemTech, Louisville, Ky.

Once the dipeptide is synthesized or purchased, it is cyclized to form adiketopiperazine. This can be accomplished by a variety of techniques.For example, U.S. Patent Application Publication Number 2004/0024180describes a method of cyclizing dipeptides. Briefly, the dipeptide isheated in an organic solvent while removing water by distillation. Insome embodiments, the organic solvent is a low-boiling azeotrope withwater, such as acetonitrile, allyl alcohol, benzene, benzyl alcohol,n-butanol, 2-butanol, t-butanol, acetic acid butylester, carbontetrachloride, chlorobenzene chloroform, cyclohexane, 1,2-dichlorethane,diethylacetal, dimethylacetal, acetic acid ethylester, heptane,methylisobutylketone, 3-pentanol, toluene and xylene. The temperaturedepends on the reaction speed at which the cyclization takes place andon the type of azeotroping agent used. In some embodiments, the reactionis carried out at 50-200° C., in some embodiments at 80-150° C. The pHrange in which cyclization takes place can be easily determine by theperson skilled in the art. In some embodiments the pH range willadvantageously be 2-9, in some embodiments 3-7.

When one or both of the amino acids of the dipeptide has, or isderivatized to have, a carboxyl group on its side chain (e.g., asparticacid or glutamic acid), the dipeptide is cyclized as described in U.S.Pat. No. 6,555,543. Briefly, the dipeptide, with the side-chain carboxylstill protected, is heated under neutral conditions. Typically, thedipeptide will be heated at from about 80° C. to about 180° C., in someembodiments at about 120° C. The solvent will be a neutral solvent. Forinstance, the solvent may comprise an alcohol (such as butanol,methanol, ethanol, and higher alcohols, but not phenol) and anazeotropic co-solvent (such as toluene, benzene, or xylene). In someembodiments, the alcohol is butan-2-ol, and the azeotropic co-solvent istoluene. The heating is continued until the reaction is complete, andsuch times can be determined empirically. Typically, the dipeptide willbe cyclized by refluxing it for about 8-24 hours, in some embodimentsabout 18 hours. Finally, the protecting group is removed from thediketopiperazine. In doing so, the use of strong acids (mineral acids,such as sulfuric or hydrochloric acids), strong bases (alkaline bases,such as potassium hydroxide or sodium hydroxide), and strong reducingagents (e.g., lithium aluminum hydride) should be avoided, in order tomaintain the chirality of the final compound.

Dipeptides made on solid phase resins can be cyclized and released fromthe resin in one step. See, e.g., U.S. Pat. No. 5,817,751. For instance,the resin having an N-alkylated dipeptide attached is suspended intoluene or toluene/ethanol in the presence of acetic acid (e.g., 1%) ortriethylamine (e.g., 4%). Typically, basic cyclization conditions areutilized for their faster cyclization times.

Other methods of cyclizing dipeptides and of making diketopiperazinesare known in the art and can be used in the preparation ofdiketopiperazines useful in the practice of the present disclosure. See,e.g., those references listed above. In addition, many diketopiperazinessuitable for use in the present disclosure can be made as describedbelow from proteins and peptides. Further, diketopiperazines for use inthe practice of the present disclosure can be obtained commerciallyfrom, e.g., DMI Synthesis Ltd., Cardiff, UK (custom synthesis).

The DA-DKP composition and/or products of the present disclosure can beprepared from solutions containing DA-DKP, including from thecommercially-available pharmaceutical compositions comprising albumin,such as human serum albumin, by well known methods, such asultrafiltration, chromatography, size-exclusion chromatography (e.g.,Centricon filtration), affinity chromatography (e.g., using a column ofbeads having attached thereto an antibody or antibodies directed to thedesired diketopiperazine(s) or an antibody or antibodies directed to thetruncated protein or peptide), anion exchange or cation exchange,sucrose gradient centrifugation, salt precipitation, or sonication, thatwill remove some or all of the albumin in the solution. The resultantDA-DKP-containing composition and/or product can be used andincorporated into pharmaceutical compositions as described above.

Using an ultrafiltration separation method, a human serum albumincomposition can be passed over an ultrafiltration membrane having amolecular weight cut-off that retains the albumin while the DA-DKPpasses into the resulting filtrate or fraction. This filtrate maycomprise components having molecular weights less than about 50 kDa,less than about 40 kDa, less than 30 kDa, less than about 20 kDa, lessthan about 10 kDa, less than about 5 kDa, less than about 3 kDa. In someembodiments, the filtrate comprises components having molecular weightsless than about 5 kDa (also referred to as “<5000 MW” and/or lowmolecular weight fraction “LMWF”). This <5000 MW fraction or filtratecontains DA-DKP which is formed after the dipeptide aspartate-alanine iscleaved from albumin and subsequently cyclized into thediketopiperazine.

The term “LMWF” refers to a low molecular weight fraction of HSA that isa composition prepared by separation of high molecular weight componentsfrom human serum albumin (HSA). For example, LMWF can be prepared byfiltration of a commercially available HSA solution wherein molecularweight components of more than 3 kilo daltons (kDa), 5 kDa, 10 kDa, 20kDa, 30 kDa, 40 kDa, 50 kDa, are separated from the HSA solution.Alternatively, the term LMWF can refer to a composition prepared byseparation of the high molecular weight components by other techniques,including but not limited to ultrafiltration, column chromatographyincluding size exclusion chromatography, affinity chromatography, anionexchange, cation exchange, sucrose gradient centrifugation, saltprecipitation, or sonication. LMWF also refers to a composition thatincludes components of HSA having a molecular weight less than 50,000daltons (Da) (or 50 kDa), 40 kDa, 30 kDa, 20 kDa, 10 kDa, 5000 Da, 4000Da, or 3000 Da (corresponding to 50,000 g/mol, 40,000 g/ml, 30,000g/mol, 20.00 g/mol, 10,000 g/mol, 5,000 g/mol, 4,000 g/mol or 3,000g/mol respectively). AMPION® (Ampio Pharmaceuticals, Inc., Englewood,Colo. USA) is a <5 kDa fraction of human serum albumin (HSA). AMPION®can be produced, for example, by filtering commercially available HSA asdescribed herein. Commercially available HSA is produced byfractionation of blood, for example by the Cohn process or itsvariations. AMPION® can be produced by filtering such commerciallyavailable HSA, for example a 5% HSA solution, to remove components above5 kDA.

AMPION® is currently approved for clinical use by the United States Foodand Drug Administration, as an anti-inflammatory, immunomodulating drug.The novel mode of action of AMPION® involves multiple biochemicalpathways associated with resolving inflammation. AMPION® is a suitabledrug for the treatment of viral diseases for several reasons, includingbut not limited to those below.

First, an important aspect of ARDS, triggered by COVID-19, is an initialrelease of chemical signals and other inflammatory mediators secreted bylung epithelial and endothelial cells. Neutrophils and someT-lymphocytes migrate into the inflamed lung tissue and contribute tothe amplification/deterioration of ARDS. A decrease in the production oflipid mediators of inflammation (prostaglandins) may impair theresolution of inflammation associated with ARDS.

Second, AMPION® was reported to up regulate the production of thesehealing lipid mediators' prostaglandins in-vitro. Gao et al., J.Immunol. (2017); 199:2043-2054. In addition, multiple inflammatorysignals (i.e., TNFα, IL6, CXCL10) have been reported to be attenuated byAMPION®, including a decrease in vascular permeability. Bar-Or et al.,On the Mechanisms of Action of the Low Molecular Weight Fraction ofCommercial Human Serum Albumin in Osteoarthritis. Current RheumatologyReviews (2019), 15, 189-200.

Third, more than 1,000 patients with another inflammatory disease,osteoarthritis, have received AMPION® without any serious drug relatedadverse events. A subset of these patients were given AMPION® multipletimes over the course of a year, and no serious drug related adverseevents were observed.

Fourth, AMPION® is formulated as a sterile liquid and is easilyadministered as a nebulized liquid form or intravenously. AMPION®,particularly in aerosolized and/or nebulized form, can be used in thetreatment of viral diseases and related conditions. For example, easinguse for treatment of Acute Respiratory Distress Syndrome (“ARDS”),AMPION® is an aqueous solution that may be delivered throughnebulization and would be suitable for suppressing inflammation in thelungs.

Physiologically-acceptable salts of the DA-DKP of the present disclosuremay also be used in the practice of the present disclosure.Physiologically-acceptable salts include conventional non-toxic salts,such as salts derived from inorganic acids (such as hydrochloric,hydrobromic, sulfuric, phosphoric, nitric, and the like), organic acids(such as acetic, propionic, succinic, glycolic, stearic, lactic, malic,tartaric, citric, glutamic, aspartic, benzoic, salicylic, oxalic,ascorbic acid, and the like) or bases (such as the hydroxide, carbonateor bicarbonate of a pharmaceutically-acceptable metal cation or organiccations derived from N,N-dibenzylethylenediamine, D-glucosamine, orethylenediamine). The salts are prepared in a conventional manner, e.g.,by neutralizing the free base form of the compound with an acid.

Additional objects, advantages and novel features of the presentdisclosure will become apparent to those skilled in the art byconsideration of the following non-limiting examples. The followingexperimental results are provided for purposes of illustration and arenot intended to limit the scope of the claims.

EXAMPLES Example 1. A Randomized, Controlled Study to Evaluate theSafety and Efficacy of AMPION® in Patients with Prolonged RespiratoryComplications after COVID-19 Infection

This study focuses on patients who have long-term symptoms and clinicalsigns related to continued respiratory illness after the viral infectionis cleared.

AMPION® is delivered by inhalation using a nebulizer as a 32 cubiccentimeters (cc) daily dose administered four times a day (8 cc pertreatment) for five days, for a total dose of a total combined dose of160 cc. This treatment regimen provides a positive clinical outcomewhile minimizing safety risks.

AMPION® is aerosolized for inhalation using the AEROGEN® Ultra handheldnebulizer (Aerogen Limited, Galway, Ireland, FDA 510K K133360), whichconsists of the AEROGEN® Pro-X Controller and the AEROGEN® Ultrahandheld unit. The system is a portable medical device for singlepatient use indicated for aerosolization of physician-prescribedsolutions for inhalation. AMPION® is nebulized using the continuoussetting on the AEROGEN® controller, and the study drug is nebulizeduntil it is fully aerosolized.

This is a randomized, controlled Phase 1 trial to evaluate the safetyand efficacy of a 5-day AMPION® inhalation treatment in participantswith prolonged respiratory complications after a COVID-19 infection.Participants (n=40) are randomized in active (n=20) or control (n=20)groups. Both groups receive the standard of care (SOC) for individualswith prolonged COVID-19 symptoms. The treatment arm receives AMPION®inhalation treatment in addition to the SOC, and the control arms willreceive the SOC alone.

The trial objectives are to evaluate the safety and tolerability ofinhaled AMPION® in adult participants with prolonged respiratorycomplications after COVID-19 infection and to assess the effect ofinhaled AMPION® compared to SOC on the clinical outcomes forparticipants with prolonged respiratory complications after a COVID-19infection. The effect of AMPION® compared to SOC on safety is determinedby assessing incidence of adverse events (AEs) and serious adverseevents (SAEs) from baseline through Day 28. The effect of AMPION®compared to SOC on respiratory symptom improvement is assessed by timeto respiratory symptom improvement as measured using the symptomquestionnaire from baseline through Day 7 and the percentage ofparticipants demonstrating improvement on the respiratory symptomquestionnaire on Days 1 through 7. The effect of AMPION® compared to SOCon respiratory symptom resolution is assessed by time to respiratorysymptom resolution as described using the symptom questionnaire frombaseline through Day 7 and change in the respiratory symptomquestionnaire from baseline through Day 7. The effect of AMPION®compared to SOC on pulmonary function is assessed by change in bloodoxygen saturation from baseline through Day 7, change in six-minute walktest score from baseline through Day 7, change in pulmonary functiontests (spirometry, lung volume, diffusion capacity) from baselinethrough Day 7, and change in chest x-ray imaging from baseline to Day28.

Diagnosis and Main Criteria for Inclusion:

1. Male or female adults: ≥18 years2. Prior diagnosis with COVID-19, as evaluated by PCR test confirminginfection, or suspected COVID-19 diagnosis based on radiologicalclinical findings.3. Two negative COVID-19 tests to indicate infection has cleared.4. Experiencing two or more COVID-19 respiratory symptoms for at least 4weeks (28 days) after initial positive COVID-19 diagnosis, includingcough, sore throat, stuffy or runny nose, shortness of breath(difficulty breathing), tightness of chest, chest discomfort, and lowexercise tolerance5. No clinical signs indicative of severe or critical COVID-19,including respiratory failure, shock, multi-organ failure.6. No clinically significant findings via electrocardiogram (ECG),including acute myocardial infarction, acute ischemic changes, atrialfibrillation, atrial flutter, paced rhythms in individuals who haveundergone permanent pacemaker placement, evidence of prior infarction,unchanged stable conduction abnormalities e.g., right bundle branchblock, or any other finding which does not significantly impactmortality.7. Women of childbearing potential and their partner must agree to useat least one highly effective method of contraception (e.g., hormonalcontraceptives [implants, injectables, combination oral contraceptives,transdermal patches, or contraceptive rings], intrauterine devices,bilateral tubal occlusion, or sexual abstinence) for the duration of thestudy.8. Informed consent obtained from the patient or the patient's legalrepresentative.

Main Criteria for Exclusion:

1. Severe or critical COVID-19 with clinical diagnosis of respiratoryfailure, pneumonia, or acute respiratory distress syndrome (ARDS).2. Patient has severe chronic obstructive or restrictive pulmonarydisease (COPD) as defined by prior pulmonary function tests, chronicrenal failure, or significant liver abnormality (e.g., cirrhosis,transplant, etc.).3. Patient is on chronic immunosuppressive medication.4. Patient requires surgery that could be life-threatening within thestudy window.5. A history of allergic reactions to human albumin (reaction tonon-human albumin such as egg albumin is not an exclusion criterion) orexcipients in 5% human albumin (N-acetyltryptophan, sodium caprylate).6. Patient has known pregnancy or is currently breastfeeding.7. Participation in another clinical trial for an investigationaltreatment for COVID-19.8. No clinically significant findings via electrocardiogram (ECG),including acute myocardial infarction, acute ischemic changes, atrialfibrillation, atrial flutter, paced rhythms in individuals who haveundergone permanent pacemaker placement, evidence of prior infarction,unchanged stable conduction abnormalities e.g., right bundle branchblock, or any other finding which does not significantly impactmortality.9. As a result of the medical review and screening investigation, thePrincipal Investigator considers the patient unfit for the study.

Safety is assessed by recording adverse events, vital signs, bloodoxygen saturation, and laboratory findings (biochemistry, hematology)for the duration of treatment and for the length of the study (28 days).

Efficacy is assessed by recording the effects of IV AMPION® compared toSOC on the clinical outcomes for participants with prolonged respiratorycomplications after a COVID-19 infection using the following clinicaloutcomes: blood oxygen saturation, COVID-19 respiratory symptomassessment, a walk test, pulmonary function tests (e.g., spirometry,lung volume, and diffusion capacity), and chest x-ray. Assessments forclinical outcomes are performed from baseline through Day 7.

Description of Study Visits Screening (−3 Days to Day 0)

Evaluate all inclusion and exclusion criteria to ensure that patientsmeet all inclusion criteria and none of the exclusion criteria.

Confirm date of initial positive COVID-19 test. Confirm date(s) of twonegative follow up COVID-19 tests.

Medical history, pre-existing conditions, and comorbidities. Include thesymptom onset date for COVID-19 symptoms.

Obtain informed consent before the starting any study specificprocedures, including COVID testing.

Baseline (Day 0)

Confirm eligibility (review inclusion/exclusion criteria).

Demographics (age, sex, race, height and weight).

Randomize patient to study arm. If randomized to the active treatmentarm, start treatment within 24 hours.

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate.

SpO₂ and supplementation oxygen mode/flow rate, as applicable.

Respiratory symptom assessment and walk test.

Pulmonary function tests (PFTs): spirometry, lung volume, diffusioncapacity.

Chest x-ray imaging.

Hematology, biochemistry tests.

Concomitant medications/therapies.

AEs.

Treatment Period (Day 1 to Day 5)

Subjects enrolled in the active treatment arm will administer inhaledAMPION® through nebulization daily. These subjects will have a generalhealth check conducted via telephone or text message on Days 1, 3, 5.

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate.

SpO₂ and supplementation oxygen mode/flow rate, as applicable.

Respiratory symptom assessment and walk test.

PFTs: spirometry, lung volume, diffusion capacity.

Concomitant medications/therapies.

AEs.

Post Treatment Follow Up (Day 7)

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate.

SpO₂ and supplementation oxygen mode/flow rate, as applicable.

Respiratory symptom assessment and walk test.

Pulmonary function tests (PFTs): spirometry, lung volume, diffusioncapacity.

Hematology, biochemistry tests.

Concomitant medications/therapies.

AEs.

Post-Treatment Follow-Up (Day 28)

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate.

SpO₂ and supplementation oxygen mode/flow rate, as applicable.

Respiratory symptom assessment and walk test.

Pulmonary function tests (PFTs): spirometry, lung volume, diffusioncapacity.

Chest x-ray imaging.

Concomitant medications/therapies.

AEs.

Assessment Methods

Demographic Data: Demographic data are collected: age, gender, race,height and weight, comorbidities.Health Check: A general health check is be conducted every other dayduring the treatment period for the AMPION® treatment group.Vital Signs: Vital signs are collected daily during the treatment periodand at follow-up visits as follows: heart rate (or pulse rate), systolicBP, diastolic BP, body temperature, respiratory rate.Blood Oxygen Saturation (SpO₂): SpO₂ is collected daily during thetreatment period and at follow-up visits using a pulse-oxygen measuringdevice.Respiratory Symptom Assessment: The symptoms and the date experiencingsymptoms are recorded at every visit from baseline to Day 7. Thefollowing symptoms are evaluated: cough, sore throat, stuffy or runnynose, shortness of breath (difficulty breathing), tightness of chest,chest discomfort, low exercise tolerance. Each symptom is rated asfollows: 0 None, 1 Mild, 2 Moderate, or 3 Severe.Walk Test: A six-minute walk test is recorded at every visit frombaseline to Day 28.

-   -   1. Flat, straight corridor 30 m (100 feet) in length    -   2. Turnaround points marked with a cone    -   3. Patient should wear comfortable clothes and shoes    -   4. Patient rests in chair for at least 10 minutes prior to test        (i.e., no warm-up period)    -   5. Heart rate and pulse oxygen saturation (SpO₂) should be        monitored throughout the test    -   6. If the patient is using supplemental oxygen, record the flow        rate and type of device    -   7. Have patient stand and rate baseline dyspnea and overall        fatigue using Borg scale (Borg G A. Psychophysical bases of        perceived exertion. Med Sci Sports Exerc 1982; 14:377).    -   8. Set lap counter to zero and timer to six minutes    -   9. Instruct the patient: Remember that the object is to walk AS        FAR AS POSSIBLE for 6 minutes, but don't run or jog. Pivot        briskly around the cone.    -   10. Standardized encouragement statements should be provided at        one minute intervals, such as “You are doing well. You        have_minutes to go” and “Keep up the good work. You have_minutes        to go.”    -   11. At the end of the test, mark the spot where the patient        stopped on the floor    -   12. If using a pulse oximeter, measure the pulse rate and SpO₂        and record    -   13. After the test record the Borg dyspnea and fatigue levels    -   14. Ask, “What, if anything, kept you from walking farther?”    -   15. Calculate the distance walked and record    -   American Thoracic Society. ATS statement: Guidelines for the        six-minute walk test. Am J Respir Crit Care Med 2002; 166:111.    -   Holland A E, Spruit M A, Troosters T, et al. An official        European Respiratory Society/American Thoracic Society technical        standard: field walking tests in chronic respiratory disease.        Eur Respir J 2014; 44:1428.        Pulmonary Function Tests (PFTs): PFTs are recorded at every        visit from baseline to Day 7 or 28 using spirometry, lung        volume, and diffusion capacity tests.        Chest X-Ray Imaging: Chest x-ray images are taken at baseline        and Day 28. The standard chest examination consists of a PA        (posterioranterior) and lateral chest x-ray. The films are read        together. The PA exam is viewed as if the patient is standing in        front of you with their right side on your left. The patient is        facing towards the left on the lateral view.        Hematology: Hematology lab tests are collected at baseline and        on Day 7. In the case of an abnormal lab result, continue to        collect and test those samples to follow subject through        resolution. The following hematology labs are tested: white cell        count, red blood cell count, hemoglobin, hematocrit, mean cell        volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin        concentration (MCHC), platelets, neutrophils, lymphocytes,        monocytes, eosinophils, and basophils.        Serum Biochemistry: Biochemistry lab tests are collected at        baseline and on Day 7. In the case of an abnormal lab result,        continue to collect and test those samples to follow subject        through resolution. The following biochemistry lab tests are        tested: sodium, potassium, chloride, bicarbonate, urea,        creatinine, glucose, total calcium, phosphate, ferritin,        high-sensitivity C-reactive protein (hs-CRP), protein, albumin,        globulins, total bilirubin, alkaline phosphatase (ALP), alanine        aminotransferase (ALT), aspartate aminotransferase (AST) and        lactate dehydrogenase (LDH).        Concomitant Medications: Concomitant medications are collected:        prior (pre-hospitalization) concomitant medications, in-patient        concomitant medications.        Adverse Events: Any documented adverse event, which is any        undesired medical occurrence in a patient or clinical        investigation patient receiving a pharmaceutical product which        does not necessarily have a causal relationship with this        treatment. These include but are not limited to the following:        cardiac injury, arrhythmia, septic shock, liver dysfunction,        acute kidney injury, and multi-organ failure.

Example 2. A Randomized, Double-Blinded, Placebo-Controlled Phase IIStudy to Evaluate the Safety and Efficacy of Inhaled AMPION® in Adultswith Respiratory Distress Secondary Due to COVID-19

This is a phase II randomized controlled trial (RCT) to evaluate inhaledAMPION® for adults with respiratory distress due to COVID-19. AMPION® isan immunomodulatory therapy with anti-inflammatory effects potentiallytreating COVID-19 patients with respiratory complications who have aneed for supplemental oxygen and breathing assistance. AMPION® targetsand reduces the production of inflammatory cytokines induced by viruses,including SARS-CoV-2, and mitigates the severity of the disease inpatients, such as those for whom ongoing inflammation is responsible forCOVID-19 disease severity and the progression to respiratory distress.

The primary trial objective is to evaluate the effect of AMPION® onall-cause mortality in adult participants with respiratory distress dueto COVID-19. The secondary trial objectives are to evaluate the safetyand efficacy of inhaled AMPION® versus control in improving the clinicalcourse and outcomes of participants with respiratory distress due toCOVID-19.

The primary endpoint assesses the effect of AMPION® compared to placeboon all-cause mortality. All-cause mortality is measured as thepercentage of participants with a successful outcome (life) orunsuccessful outcome (death) by Day 28.

Secondary endpoints assess the effect of inhaled AMPION® treatmentcompared to placebo on the clinical outcomes for participants withrespiratory distress COVID-19. The effect of AMPION® compared to placeboon safety is assessed as incidence of adverse events (AEs) and seriousadverse events (SAEs) from baseline at Day 5. The effect of AMPION®compared to placebo on hospital stay is assessed as hospital length ofstay (LOS) from admission to discharge. The effect of AMPION® comparedto placebo on oxygen use is assessed as duration of oxygen use, bloodoxygen saturation, and oxygen flow rate from baseline through Day 5. Theeffect of AMPION® compared to placebo on progression to respiratoryfailure is assessed as proportion of participants who progress torespiratory failure (i.e., need for mechanical ventilation, ECMO,non-invasive ventilation) by Day 28. The effect of AMPION® compared toplacebo on intensive care is assessed as percentage of participants whorequire Intensive Care Unit (ICU) admission by Day 28 and as ICU LOSfrom ICU admission to discharge. The effect of AMPION® compared toplacebo on clinical improvement is assessed as change in ordinal scalefrom baseline through Day 5 and as change in ordinal scale from baselineto hospital discharge.

Exploratory endpoints assess the effect of inhaled AMPION® treatmentcompared to placebo on the clinical outcomes for participants withrespiratory distress due to COVID-19. The effect of AMPION® compared toplacebo on clinical health is assessed as change in NEWS2 score frombaseline through Day 5 and as change in NEWS2 score from baseline tohospital discharge. The effect of AMPION® compared to placebo cytokineprofile is assessed as modulation of cytokine levels from baseline toDay 5.

Inclusion Criteria—Patients should Fulfill all the Following InclusionCriteria:1. Male or female, ≥18 years old2. Diagnosed with COVID-19, as evaluated by laboratory diagnostic testor diagnosis based on radiological clinical findings.3. Respiratory distress as evidenced by at least two of the following:

Radiographic infiltrates by imaging (chest x-ray, CT scan, etc.)

Recording of SpO₂≤90% or the patient is requiring oxygen to maintain anSpO₂≥90%.

Requiring supplemental oxygen.

Diagnosis of mild, moderate, or severe ARDS by Berlin definition.

4. Informed consent obtained from the patient or the patient's legalrepresentative.

Exclusion Criteria—Patients Fulfilling One or More of the FollowingCriteria May not be Enrolled in the Study:

1. As a result of the medical review and screening investigation, thePrincipal Investigator considers the patient unfit for the study and/orprogression to death is imminent and inevitable irrespective of theprovision of treatments.2. Patient has severe chronic obstructive or restrictive pulmonarydisease (COPD) (as defined by prior pulmonary function tests), chronicrenal failure, or significant liver abnormality (e.g., cirrhosis,transplant, etc.).3. Patient has chronic conditions requiring chemotherapy orimmunosuppressive medication.4. A history of allergic reactions to human albumin (reaction tonon-human albumin such as egg albumin is not an exclusion criterion) oringredients in 5% human albumin (N-acetyltryptophan, sodium caprylate).5. Prolonged QT interval.6. Patient has known pregnancy or is currently breastfeeding.7. Patient planning to become pregnant, or father a child, during thetreatment and follow-up period and/or is not willing to remain abstinentor use contraception.8. Participation in another clinical trial (not including treatments forCOVID-19 as approved by the FDA through expanded access, emergency, orcompassionate use), or participation in a trial in the last 30 days.

Participants are randomized 1:1 to one of two groups, active (AMPION®)or control (placebo). Participants randomized to the active arm (n=100)will receive a daily dose (32 mL/day) of AMPION® inhaled via nebulizerdelivered in four equally divided doses of 8 mL, every 6 hours.Participants randomized to the control arm (n=100) will receive a dailydose (32 mL/day) of placebo inhaled via nebulizer delivered in fourequally divided doses of 8 mL, every 6 hours. Treatment is repeated for5 days.

Nebulized study intervention (active or placebo) is delivered using theAEROGEN® Solo Nebulizer System with the AEROGEN® Solo Adaptor (FDA 510KK133360) manufactured by AEROGEN® Limited, Galway, Ireland. The AEROGEN®Solo Adaptor is a vibrating mesh nebulizer with a drug reservoir used ina hospital setting for delivery of respiratory therapy, including thehand-held AEROGEN® Ultra, NIV, and/or a mechanically ventilated circuit.

All participants receive the SOC for COVID-19 in a hospital setting asrequired based on disease severity, or as required for the course ofhospital stay as follows:

Oxygen administration to maintain oxygen saturation of 90% or greater,including the use of supplemental oxygen, NIV, and mechanicalventilation circuits.

Nursing physical that may include review of neurological; pulmonary;cardiac; gastrointestinal; and urinary assessment at least daily duringtreatment.

Vital monitoring (heart rate, blood pressure, temperature, respiratoryrate, SpO₂) at least daily during treatment.

Telemetry monitoring to evaluate heart rhythm and rate.

Diet as tolerated to satisfy nutritional needs.

Treatments for COVID-19 symptoms including antibiotics, coughsuppressants/expectorants, anti-coagulants, fever reducers/pain killers,anti-nausea drugs, and/or bronchodilators.

Treatments for COVID-19 as approved by the FDA including expandedaccess, emergency, or compassionate use (i.e., remdesivir,dexamethasone, convalescent plasma).

Medications are recorded as concomitant medication, tabulated, andcompared among groups.

Primary efficacy is assessed by recording the effects of AMPION®compared to placebo on mortality. Secondary endpoints will evaluatesafety and other clinical outcomes of AMPION® compared to placebo onlength of stay (LOS) in the hospital, oxygen use (including SpO₂ andflow rate), progression to respiratory failure (i.e., need formechanical ventilation, ECMO, non-invasive ventilation), need forintensive care, ICU LOS, and ordinal scale for clinical improvement(8-point scale). Exploratory endpoints will evaluate safety and otherclinical outcomes of AMPION® compared to placebo National Early WarningScore (NEWS) 2 score for the degree of illness of a patient) andmodulation of cytokine levels.

Safety is assessed by recording adverse events, vital signs, bloodoxygen saturation, and laboratory findings (biochemistry, hematology)for the duration of treatment and for the length of stay in the hospital(as applicable). Laboratory tests (biochemistry, hematology) areperformed at baseline and every other day through treatment and throughhospital stay unless an abnormal value is observed. In the case of anabnormal lab result, continue to collect and test those samples tofollow subject through resolution.

Description of Study Visits Screening (−3 Days to Day 0)

Evaluate all inclusion and exclusion criteria to ensure that patientsmeet all inclusion criteria and none of the exclusion criteria.

Medical history, pre-existing conditions, and comorbidities. Include thesymptom onset date for COVID-19 symptoms and date of COVID-19 test.

Diphenhydramine, hydroxychloroquine, and azithromycin all prolong thecardiac QT interval, increasing risk of fatal cardiac arrhythmia.Therefore, severely-ill subjects receiving multiple drugs that prolongQT intervals are reviewed carefully by the P.I. on a case-by-caseadjudication for benefit-risk ratio and close cardiovascular monitoring.Note, subjects who have baseline QT prolongation are excluded from thisstudy.

Obtain informed consent before the starting any study specificprocedures, including COVID testing.

Baseline (Day 0)

Confirm eligibility (review inclusion/exclusion criteria).

Randomize patient to study arm.

Demographics (age, sex, race, height and weight)

Concomitant medications/therapies

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate.

SpO₂ and supplementation oxygen mode/flow rate

ECG monitoring (telemetry) or 12-lead ECG: record aberrant changes inwaves/intervals

Hematology, biochemistry tests

Cytokine and chemokine assays

Ordinal scale and NEWS2 assessments

AEs

Treatment Period (Day 1 to Day 5)

Begin inhalation treatment of study intervention (active or placebo)within 6 hours of randomization.

Concomitant medications/therapies

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate

SpO₂ and supplementation oxygen mode/flow rate

ECG monitoring (telemetry) or 12-lead ECG: record aberrant changes inwaves/intervals—monitored as needed for patients who have abnormalreadings or events requiring measurements.

Hematology, biochemistry tests—these tests are performed at baseline andevery other day through treatment and through hospital stay unless anabnormal value is observed. In the case of an abnormal lab result,continue to collect and test those samples to follow subject throughresolution.

Cytokine and chemokine assays—these tests are performed at baseline andat Day 5.

Ordinal scale and NEWS2 assessments

AEs

Hospitalization Period, as Applicable (Day 6 Through Hospital Discharge)

Concomitant medications/therapies

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate

SpO₂ and supplementation oxygen mode/flow rate

ECG monitoring (telemetry) or 12-lead ECG: record aberrant changes inwaves/intervals—monitored as needed for patients who have abnormalreadings or events requiring measurements.

Hematology, biochemistry tests—these tests are performed at baseline andevery other day through treatment and through hospital stay unless anabnormal value is observed. In the case of an abnormal lab result,continue to collect and test those samples to follow subject throughresolution.

Cytokine and chemokine assays—these tests are performed at hospitaldischarge.

Ordinal scale and NEWS2 assessments

AEs

Post-Treatment Follow-Up (Days 28, 60)

Mortality

Concomitant medications/therapies

Hospital LOS

ICU LOS

Ordinal scale and NEWS2 assessments

AEs

Assessment Methods

Demographic Data: Demographic data are collected from medical records:age, gender, race, height and weight, comorbidities.Medical History: Medical history and pre-existing conditions arecollected from medical records.Concomitant Medications: Concomitant medications are collected from themedical records: prior (pre-hospitalization) concomitant medications,in-patient concomitant medications.Mortality: All-cause mortality is recorded at hospital discharge, Days28 and 60, as applicable. Cause of mortality is assessed and documented.All-cause mortality is calculated for the primary endpoint as thepercentage of participants with a successful outcome (life) orunsuccessful outcome (death).Length of Stay (LOS): Dates of hospitalization and ICU admission as wellas discharge dates are recorded at hospital discharge, Days 28 and 60,as applicable. The hospital LOS and ICU LOS are calculated as follows:

ICU admission: defined (in days) as the first study day when ICU statusis changed to “yes” minus baseline date+1

Hospital LOS: is defined (in days) as the date of hospital dischargeminus date of hospital admission+1

ICU LOS: defined (in days) as the date moved out of ICU minus firststudy date when ICU admission changed to “yes”+1

Oxygen Use: Oxygen use measured as blood oxygen saturation (SpO₂) andoxygen flow rate (liters per minute, 1 pm) are recorded at every visitfrom baseline to Day 5.Intubation/Extubation: Date and time of intubation/extubation and dayson ventilator are recorded at hospital discharge, Days 28 and 60, asapplicable. Proportion of participants who progress to respiratoryfailure (i.e., need for mechanical ventilation, ECMO, non-invasiveventilation, or high-flow nasal cannula oxygen) are evaluated.Vital Signs: Vital signs are collected daily during the treatment periodand at follow-up visits from medical records as follows: heart rate (orpulse rate), systolic BP, diastolic BP, body temperature (° F. in theUS, ° C. out of the US), respiratory rate.Hematology: Hematology lab tests are collected from the medical recordsat baseline and every other day through treatment and hospital stay (asapplicable) unless an abnormal value is observed. In the case of anabnormal lab result, continue to collect and test those samples tofollow subject through resolution. The following hematology labs aretested: white cell count, red blood cell count, hemoglobin, hematocrit,mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobinconcentration (MCHC), platelets, neutrophils, lymphocytes, monocytes,eosinophils, and basophils.Serum Biochemistry: Biochemistry lab tests are collected from themedical records at baseline and every other day through treatment andhospital stay (as applicable) unless an abnormal value is observed. Inthe case of an abnormal lab result, continue to collect and test thosesamples to follow subject through resolution. The following biochemistrylab tests are tested: sodium, potassium, chloride, bicarbonate, urea,creatinine, glucose, total calcium, phosphate, ferritin,high-sensitivity C-reactive protein (hs-CRP), protein, albumin,globulins, total bilirubin, alkaline phosphatase (ALP), alanineaminotransferase (ALT), aspartate aminotransferase (AST) and lactatedehydrogenase (LDH).Cytokine and Chemokine Assays: Cytokine and chemokine assay lab testresults are collected and recorded before and after treatment (atbaseline and again at Day 5 and at hospital discharge). A cytokine panelis tested to include, but not limited to: Tumor Necrosis Factor alpha(TNFα), interferon gamma (IFNγ), Interleukin 1 beta (IL-1β),interleukins (IL-6, IL-8, IL-10, IL-12). Data is reported for researchpurposes, and the sample is destroyed after data is collected.Ordinal Scale for Clinical Improvement: The clinical status is recordedat every visit from baseline to Day 60 using the World HealthOrganization (WHO) “Ordinal Scale for Clinical Improvement”. This8-point ordinal scale was created to be responsive to the eligiblepatient population, intervention, and course of illness of COVID-19. Thefollowing scores are collected:

Score Descriptor 0 No clinical or virological evidence of infection 1 Nolimitation of activities 2 Limitation of activities 3 Hospitalized, nooxygen 4 Hospitalized, oxygen by mask or nasal prongs 5 Hospitalized,non-invasive ventilation or high-flow oxygen 6 Hospitalized, mechanicalventilation 7 Hospitalized, ventilation + additional organ support —pressors, RRT, ECMO 8 DeathNEWS2 Score for Determining the Degree of Illness: The National EarlyWarning Score 2 (NEWS2) score, a well-known clinical risk score, isrecorded at every visit from baseline to Day 60 using the vitalscollected during the visit. The NEWS2 score is recommended by theNational Institute for Health and Care Excellence (“NICE”) for managingCOVID-19 patients.Adverse Events: Any documented adverse event, which is any undesiredmedical occurrence in a patient or clinical investigation patientreceiving a pharmaceutical product which does not necessarily have acausal relationship with this treatment. These include but are notlimited to the following: cardiac injury, arrhythmia, septic shock,liver dysfunction, acute kidney injury, and multi-organ failure.

Results

27 patients have been enrolled in the study. The mean age of patients is67 years, with 63% male and 93% white patients. Dexamethasone wasadministered for nearly all subjects (n=23), and REMDESIVIR® was used in10 subjects. All patients have completed their 5-day treatment window,with no serious safety observations to date.

The study indicates positive clinical outcomes due to AMPION® use inCOVID-19 patients with respiratory distress. The all-cause mortality inCOVID-19 patients was lower in the AMPION® treatment arm compared tocontrol (7.7% AMPION®; 21.4% SOC; n=27). Following 5-day treatment, itwas observed that subjects who received AMPION® may require less oxygenthan SOC patients (average oxygen use at 5 liters per minute, LPM, forAMPION®; 9 LPM for SOC) with a greater proportion of AMPION®-treatedsubjects remaining stable, or decreasing the amount of required oxygen,compared to SOC (86% of AMPION®; 75% of SOC). Improvement on the WHOordinal scale were observed in AMPION® patients as early as Day 2 andcontinued to Day 5 with 86% vs 75% of patients remaining stable ordemonstrating improvement with AMPION® treatment compared to SOC,respectively, by Day 5. In subjects who have been discharged from thehospital (n=17), patients who received AMPION® required lesshospitalization time overall compared to SOC alone (6 days with AMPION®treatment; 9 days with SOC).

Example 3. A Randomized, Double-Blinded, Placebo-Controlled Phase I/IIStudy to Evaluate the Safety and Efficacy of Intravenous AMPION® inAdult COVID-19 Patients Requiring Oxygen Supplementation

There are primary endpoints for safety and efficacy. The primaryendpoint for safety is incidence and severity of AEs at Day 5. Theprimary efficacy endpoint is the effect of AMPION® on all-causemortality by Day 28.

Secondary efficacy endpoints assess the effect of IV AMPION® compared toSOC on the clinical outcomes for participants with COVID-19 who requiresupplemental oxygen. The effect of AMPION® compared to placebo onhospital stay is assessed as hospital length of stay (LOS) fromadmission to discharge. The effect of AMPION® compared to placebo onoxygen use is assessed as duration of oxygen use, blood oxygensaturation, and oxygen flow rate from baseline through Day 5. The effectof AMPION® compared to placebo on progression to respiratory failure isassessed as proportion of participants who progress to respiratoryfailure (i.e., need for mechanical ventilation, ECMO, non-invasiveventilation) by Day 28. The effect of AMPION® compared to placebo onintensive care is assessed as percentage of participants who requireIntensive Care Unit (ICU) admission by Day 28 and as ICU LOS from ICUadmission to discharge. The effect of AMPION® compared to placebo onclinical improvement is assessed as change in ordinal scale frombaseline through Day 5 and as change in ordinal scale from baseline tohospital discharge. The effect of AMPION® compared to placebo onclinical health is assessed as change in NEWS2 score from baselinethrough Day 5 and as change in NEWS2 score from baseline to hospitaldischarge. The effect of AMPION® compared to placebo on oxygen use isassessed as duration of oxygen use, blood oxygen saturation, and oxygenflow rate from baseline through Day 5.

The participant population are those infected with SARS-CoV-2 that havedeveloped severe symptoms consistent with COVID-19 and requiresupplemental oxygen to maintain blood oxygen saturation levels.Treatment of COVID-19 depends on the stage and severity of disease witha hyperinflammatory state observed in the moderate to severe stages thatare thought to lead to clinical complications, so treatment withimmunomodulators at this point in the disease is more effective thananti-viral treatments.

Inclusion Criteria

Patients should fulfill all the following inclusion criteria:1. Male or female, ≥18 years old.2. Diagnosed with COVID-19, as evaluated by laboratory diagnostic testor diagnosis based on radiological clinical findings.3. Clinical signs indicative of severe systemic illness with COVID-19,such as respiratory rate 22 30 per minute, heart rate ≥125 per minute,SpO₂≤93% on room air at sea level (SpO₂≤90% at altitude) orPaO₂/FiO_(2<300)4. Patient is receiving supplemental oxygen to maintain a SpO_(2≥90)%.5. Informed consent obtained from the patient or the patient's legalrepresentative.

Exclusion Criteria

Patients fulfilling one or more of the following criteria may not beenrolled in the study:1. As a result of the medical review and screening investigation, thePrincipal Investigator considers the patient unfit for the study and/orprogression to death is imminent and inevitable irrespective of theprovision of treatments.2. Patient has severe chronic obstructive or restrictive pulmonarydisease (COPD) (as defined by prior pulmonary function tests), chronicrenal failure, or significant liver abnormality (e.g., cirrhosis,transplant, etc.).3. Patient has chronic conditions requiring chemotherapy orimmunosuppressive medication.4. A history of allergic reactions to human albumin (reaction tonon-human albumin such as egg albumin is not an exclusion criterion) oringredients in 5% human albumin (N-acetyltryptophan, sodium caprylate).5. Prolonged QT interval.6. Patient has known pregnancy or is currently breastfeeding.7. Patient planning to become pregnant, or father a child, during thetreatment and follow-up period and/or is not willing to remain abstinentor use contraception.8. Participation in another clinical trial (not including treatments forCOVID-19 as approved by the FDA through expanded access, emergency, orcompassionate use), or participation in a trial in the last 30 days.

Study Plan

Participants randomized to the active arm (n=30) receive a daily dose(250 mL/day) of IV AMPION® delivered in two equally divided doses of 125mL, every 12 hours. Participants randomized to the control arm (n=30)receive a daily dose (250 mL/day) of IV placebo delivered in two equallydivided doses of 125 mL, every 12 hours. Treatment is repeated for 5days.

Intravenous study intervention (active or placebo) AMPION® areadministered using an infusion pump through an existing intravenousaccess (central line, PICC, peripheral, or saline lock). AMPION® isadministered as an intravenous piggyback/secondary if the patient at thetime of the administration of AMPION® has an existing primary infusion.If there are no intravenous fluids being administered at the time of theAMPION® infusion, AMPION® treatment is infused as an intermittentintravenous infusion. AMPION® is administered undiluted at a flow rateof 100 cc/hour.

All participants receive the SOC for COVID-19 in a hospital setting asrequired based on disease severity, or as required for the course ofhospital stay as follows:

Oxygen administration to maintain oxygen saturation of 90% or greater.

Nursing physical that may include review of neurological; pulmonary;cardiac; gastrointestinal; and urinary assessment at least daily duringtreatment.

Vital monitoring (heart rate, blood pressure, temperature, respiratoryrate, SpO₂) at least daily during treatment.

Telemetry monitoring to evaluate heart rhythm and rate.

Diet as tolerated to satisfy nutritional needs.

Treatments for COVID-19 symptoms including antibiotics, coughsuppressants/expectorants, anti-coagulants, fever reducers/pain killers,anti-nausea drugs, and/or bronchodilators.

Treatments for COVID-19 as approved by the FDA including expandedaccess, emergency, or compassionate use (i.e., remdesivir,dexamethasone, convalescent plasma).

Medications are recorded as concomitant medication, tabulated, andcompared among groups.

Safety is assessed by recording adverse events, vital signs, bloodoxygen saturation, and laboratory findings (biochemistry, hematology)for the duration of treatment and for the length of stay in the hospital(as applicable). Laboratory tests (biochemistry, hematology) areperformed at baseline and every other day through treatment and throughhospital stay unless an abnormal value is observed. In the case of anabnormal lab result, continue to collect and test those samples tofollow subject through resolution.

Efficacy is assessed by recording the effects of IV AMPION® compared toplacebo on the clinical outcomes for participants with COVID-19 usingthe following: mortality, oxygen use (including SpO₂ and flow rate),need for intensive care, length of stay (LOS) in the hospital, LOS inthe ICU, ordinal scale for clinical improvement (8-point scale),National Early Warning Score (NEWS) 2 score for the degree of illness ofa patient, and modulation of cytokine levels.

Description of Study Visits Screening (−3 Days to Day 0)

Evaluate all inclusion and exclusion criteria to ensure that patientsmeet all inclusion criteria and none of the exclusion criteria.

Medical history, pre-existing conditions, and comorbidities. Include thesymptom onset date for COVID-19 symptoms and date of COVID-19 test.

Diphenhydramine, hydroxychloroquine, and azithromycin all prolong thecardiac QT interval, increasing risk of fatal cardiac arrhythmia.Therefore, severely-ill subjects receiving multiple drugs that prolongQT intervals are reviewed carefully by the P.I. on a case-by-caseadjudication for benefit-risk ratio and close cardiovascular monitoring.Note, subjects who have baseline QT prolongation are excluded from thisstudy.

Obtain informed consent before the starting any study specificprocedures, including COVID testing.

Baseline (Day 0)

Confirm eligibility (review inclusion/exclusion criteria).

Randomize patient to study arm.

Demographics (age, sex, race, height and weight)

Concomitant medications/therapies

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate

SpO₂ and supplementation oxygen mode/flow rate

ECG monitoring (telemetry) or 12-lead ECG: record aberrant changes inwaves/intervals

Hematology, biochemistry tests

Cytokine and chemokine assays

Ordinal scale and NEWS2 assessments

AEs

Treatment Period (Day 1 to Day 5)

Administer AMPION® or placebo control as a daily IV infusion. Begintreatment within 6 hours of randomization.

Concomitant medications/therapies

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate—vital signs are collected shortly after (within 30minutes) treatment.

SpO₂ and supplementation oxygen mode/flow rate

ECG monitoring (telemetry) or 12-lead ECG: record aberrant changes inwaves/intervals—monitored as needed for patients who have abnormalreadings or events requiring measurements.

Hematology, biochemistry tests—these tests are performed at baseline andevery other day through treatment and through hospital stay unless anabnormal value is observed. In the case of an abnormal lab result,continue to collect and test those samples to follow subject throughresolution.

Cytokine and chemokine assays—these tests are performed at baseline andat Day 5.

Ordinal and NEWS2 assessments

AEs

Hospitalization Period, as Applicable (Day 6 Through Hospital Discharge)

Concomitant medications/therapies

Vital signs: heart rate, systolic and diastolic BP, body temperature,respiratory rate

SpO₂ and supplementation oxygen mode/flow rate

ECG monitoring (telemetry) or 12-lead ECG: record aberrant changes inwaves/intervals—monitored as needed for patients who have abnormalreadings or events requiring measurements.

Hematology, biochemistry tests—these tests are performed at baseline andevery other day through treatment and through hospital stay unless anabnormal value is observed. In the case of an abnormal lab result,continue to collect and test those samples to follow subject throughresolution.

Cytokine and chemokine assays—these tests are performed at hospitaldischarge.

Ordinal scale and NEWS2 assessments

AEs

Post-Treatment Follow-Up (Days 28, 60)

Mortality

Concomitant medications/therapies

Hospital LOS

ICU LOS

Ordinal scale and NEWS2 assessments

AEs

Assessment Methods

Demographic Data: Demographic data is collected from medical records:age, gender, race, height and weight, comorbidities.Medical History: Medical history and pre-existing conditions arecollected from medical records.Concomitant Medications: Concomitant medications are collected from themedical records: prior (pre-hospitalization) concomitant medications,in-patient concomitant medications.Mortality: All-cause mortality is recorded at hospital discharge, Days28 and 60, as applicable. Cause of mortality is assessed and documented.All-cause mortality is calculated for the primary endpoint as thepercentage of participants with a successful outcome (life) orunsuccessful outcome (death).Length of Stay (LOS): Dates of hospitalization and ICU admission as wellas discharge dates are recorded at hospital discharge, Days 28 and 60,as applicable. The hospital LOS and ICU LOS are calculated as follows:

ICU admission: defined (in days) as the first study day when ICU statusis changed to “yes” minus baseline date+1

Hospital LOS: is defined (in days) as the date of hospital dischargeminus date of hospital admission+1

ICU LOS: defined (in days) as the date moved out of ICU minus firststudy date when ICU admission changed to “yes”+1

Oxygen Use: Oxygen use measured as blood oxygen saturation (SpO₂) andoxygen flow rate (liters per minute, 1 pm) are recorded at every visitfrom baseline to Day 5.Intubation/Extubation: Date and time of intubation/extubation and dayson ventilator are recorded at hospital discharge, Days 28 and 60, asapplicable. Proportion of participants who progress to respiratoryfailure (i.e., need for mechanical ventilation, ECMO, non-invasiveventilation, or high-flow nasal cannula oxygen) is evaluated.Vital Signs: Vital signs are collected daily during the treatment periodand at follow-up visits from medical records as follows: heart rate (orpulse rate), systolic BP, diastolic BP, body temperature (° F. in theUS, ° C. out of the US), respiratory rate. Vital signs are collectedshortly after (within 30 minutes) treatment.Hematology: Hematology lab tests are collected from the medical recordsat baseline and every other day through treatment and hospital stay (asapplicable) unless an abnormal value is observed. In the case of anabnormal lab result, continue to collect and test those samples tofollow subject through resolution. The following hematology labs aretested: white cell count, red blood cell count, hemoglobin, hematocrit,mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobinconcentration (MCHC), platelets, neutrophils, lymphocytes, monocytes,eosinophils, and basophils.Serum Biochemistry: Biochemistry lab tests are collected from themedical records at baseline and every other day through treatment andhospital stay (as applicable) unless an abnormal value is observed. Inthe case of an abnormal lab result, continue to collect and test thosesamples to follow subject through resolution. The following biochemistrylab tests are tested: sodium, potassium, chloride, bicarbonate, urea,creatinine, glucose, total calcium, phosphate, ferritin,high-sensitivity C-reactive protein (hs-CRP), protein, albumin,globulins, total bilirubin, alkaline phosphatase (ALP), alanineaminotransferase (ALT), aspartate aminotransferase (AST) and lactatedehydrogenase (LDH).Cytokine and Chemokine Assays: Cytokine and chemokine assay lab testresults are collected and recorded before and after treatment (atbaseline and again at Day 5 and at hospital discharge). A cytokine panelis tested to include, but not limited to: Tumor Necrosis Factor alpha(TNFα), interferon gamma (IFNγ), Interleukin 1 beta (IL-1β),interleukins (IL-6, IL-8, IL-10, IL-12). Data is reported for researchpurposes, and the sample is destroyed after data is collected.Ordinal Scale for Clinical Improvement: The clinical status is recordedat every visit from baseline to Day 60 using the World HealthOrganization (WHO) “Ordinal Scale for Clinical Improvement”. See Example2 at Ordinal Scale for Clinical Improvement for further information onthe ordinal scale.NEWS2 Score for Determining the Degree of Illness: The National EarlyWarning Score 2 (NEWS2) score, a well-known clinical risk score, isrecorded at every visit from baseline to Day 60 using the vitalscollected during the visit. The NEWS2 score is recommended by theNational Institute for Health and Care Excellence (“NICE”) for managingCOVID-19 patients.Adverse Events: Any documented adverse event, which includes anyundesired medical occurrence in a patient or clinical investigationpatient receiving a pharmaceutical product which does not necessarilyhave a causal relationship with this treatment. These include but arenot limited to the following: cardiac injury, arrhythmia, septic shock,liver dysfunction, acute kidney injury, and multi-organ failure.

Results

The incidence of adverse events (AEs) at day 90 was determined, whichwas successfully achieved with no treatment-related serious adverseevents reported, and no difference in the incidence or severity of AEsbetween the AMPION® treatment and standard of care (SOC) at Day 5 and atthe 90-day follow-up.

Ten hospitalized patients with lab-confirmed COVID-19 infection who werereceiving supplemental oxygen were randomized 1:1 to AMPION® plus SOC(active) or SOC alone (control). The standard of care (SOC) includedrespiratory support, corticosteroids, and anti-viral therapies such asRemdesivir. The majority of patients were male (70%), white (90%), andhad a median age of 74 years.

Nearly all (90%) patients received dexamethasone: 80% AMPION® and 100%SOC. Remdesivir was not required for AMPION® treated patients (0%AMPION® vs. 80% SOC). A greater proportion of patients treated withAMPION® had clinical improvement from baseline on the WHO ordinal scaleon each day on treatment; Day 5 improvement was 75% AMPION® vs. 50% SOC.Likewise, a greater proportion of patients treated with AMPION® hadimprovements in oxygen requirements from baseline each day on treatmentcompared to SOC (Day 5 improvement: 75% AMPION® vs. 50% SOC).

Example 4. Calculation of AMPION® Dose

This example describes a method to calculate the dose of AMPION® forintravenous administration. This method can be applied to otheradministration routes.

Human serum albumin, including 5% human serum albumin (HSA), has beenadministered safely via IV for many years at volumes exceeding 500 cc ina day (5% HSA prescribing information). A comparison of the activecomponents in AMPION® against those in the starting material HSAdemonstrates that the proposed daily dose (250 cc) of AMPION® can besafely administered via IV.

A daily dose of 250 cc is based on AMPION®'s in vitro biologicalactivity and the amount of AMPION® required to achieve its in vitroanti-inflammatory effect (FIG. 1). The biological activity of AMPION®was measured using a bioassay which tests the release ofpro-inflammatory cytokine (TNFα) in activated immune cells. In thebioassay, human peripheral blood mononuclear cells (PBMC) are stimulatedto release TNFα. The cells are treated with either AMPION® or saline,and AMPION® activity is reported as inhibition (%) of TNFα in cellstreated with AMPION® compared to the cells treated with saline. In thebioassay, the treatment effect is achieved by adding 50 μL of AMPION® to100,000 cells. A dose-dependent effect is observed.

To model efficacy of an in vivo clinical dose of AMPION®, the numberblood monocytes in the bloodstream provides a means of estimating theconcentration of immune cells in circulation. Based on the averagemonocyte density in an adult human bloodstream, and the ratio of cellsused in the AMPION® TNFα bioassay, a therapeutic dose range of AMPION®(250-1187 cc) would be required to achieve the anti-inflammatory effect.

While various embodiments of the present disclosure have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present disclosure, as set forth in thefollowing exemplary claims.

What is claimed:
 1. A method of treating one or more symptoms of aSARS-Coronavirus-2 (SARS-CoV-2) infection in a patient, comprisingadministering to the patient a pharmaceutical composition prepared byremoving albumin from a solution of a human serum albumin composition.2. The method of claim 1, wherein the one or more symptoms are selectedfrom the group consisting of acute respiratory distress syndrome (ARDS),acute lung injury (ALI), interstitial lung disease, pulmonary fibrosis,pneumonia, reactive airway disease syndrome, respiratory distressrequiring supplemental oxygen, long COVID and combinations thereof. 4.The method of claim 1, wherein the one or more symptoms are selectedfrom the group consisting of fatigue, shortness of breath or difficultybreathing, low exercise tolerance, low blood oxygen saturation, cough,sore throat, stuffy or runny nose, joint pain, chest pain, tightness ordiscomfort, muscle pain, muscle weakness, fever, heart palpitations,difficulty thinking and/or concentrating, depression and combinationsthereof.
 5. The method of claim 4, wherein the patient has experiencedthe one or more symptoms for at least four weeks, at least one month, atleast two months, or at least three months.
 6. The method of claim 1,wherein the administration results in an outcome selected from the groupconsisting of reduced ventilator time, reduced mortality, improvement inoxygenation parameters, reduced time to resolution of one or morerespiratory symptoms, improved pulmonary function, and combinationsthereof.
 7. The method of claim 1, wherein, after the administration,the patient achieves improvement on the World Health OrganizationCOVID-19 ordinal scale of at least 4, at least 3, at least 2, or atleast
 1. 8. The method of claim 1, wherein the composition isadministered in a form suitable for administration to the lungs.
 9. Themethod of claim 8, wherein the composition is administered in anebulized form at a dose of 8 milliliters, quater in die, for five days.10. The method of claim 1, wherein the composition comprises DA-DKP. 11.The method of claim 10, wherein the composition further comprisesN-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinationsthereof.
 12. A method of treating inflammation associated with aSARS-Coronavirus-2 (SARS-CoV-2) infection in a patient, comprisingadministering to the patient a pharmaceutical composition prepared byremoving albumin from a solution of a human serum albumin composition.13. The method of claim 12, wherein the inflammation is of a tissueselected from the group consisting of lung, brain, heart, kidney, bloodvessel, skin, nerve, and combinations thereof.
 14. The method of claim12, wherein the inflammation causes a symptom selected from the groupconsisting of fatigue, shortness of breath or difficulty breathing, lowexercise tolerance, low blood oxygen saturation, cough, sore throat,stuffy or runny nose, joint pain, chest pain tightness or discomfort,muscle pain, muscle weakness, fever, heart palpitations, difficultythinking and/or concentrating, depression, and combinations thereof. 15.The method of claim 14, wherein the patient has experienced the symptomfor at least four weeks, at least one month, at least two months, or atleast three months.
 16. The method of claim 12, wherein administrationresults in an outcome selected from the group consisting of reducedventilator time, reduced mortality, improvement in oxygenationparameters, reduced time to resolution of one or more respiratorysymptoms, improved pulmonary function, and combinations thereof.
 17. Themethod of claim 12, wherein, after the administration, the patientachieves improvement on the World Health Organization COVID-19 ordinalscale of at least 4, at least 3, at least 2, or at least
 1. 18. Themethod of claim 12, wherein the patient has or had respiratory distressrequiring supplemental oxygen caused by SARS-Coronavirus-2 (SARS-CoV-2)infection.
 19. The method of claim 12, wherein the composition isadministered in a form suitable for administration to the lungs.
 20. Themethod of claim 19, wherein the composition is administered in anebulized form at a dose of 8 milliliters quater in die for five days.